Project Summary Despite advances in the field of transplantation that have curbed acute rejection through immunosuppressive drugs and better control of infection and ischemia-reperfusion injury, chronic allograft rejection is still a major obstacle for successful organ transplantation. Successful organ transplantation appears to require a balanced function of effector and regulatory T cells (Treg) to prevent the emergence of Th17 based fibrosis and fibro-obliterative processes in the allograft. Th17 cells have been strongly associated with autoimmune disease, including lupus, rheumatoid arthritis, psoriasis and multiple sclerosis, in addition, to its key role in the chronic rejection of lung and heart transplants. An assay that can quantitate potential Treg-Th17 suppression in an antigen-specific manner would be invaluable in predicting/monitoring the development and maintenance of tolerance that is perturbed in chronic inflammatory-auto-immune pathologies such as chronic rejection of allograft. Existing animal transplantation models and the trans-vivo delayed type hypersensitivity (tvDTH) assay have provided tremendous insight into tolerance mechanisms. Still, two big hurdles keep these experimental approaches from achieving commercial and clinical implementation: 1) The use of an animal model (tvDTH and transplantation), 2) The high number of cells required for the assay (tvDTH). The first hurdle is addressed by the development of the T-Cell Based Cytokine assay (T-CBC) at the University of Wisconsin. This assay detects tolerogenic factors, including IL-35, CD39 and TGF? in cultures of human PBMCs. Preliminary data from the University of Wisconsin suggests that the level of tolerogen (e.g., Ebi3(IL35), CD39 and TGF-?) signaling between Tregs and other immune cells strongly correlates with tvDTH endpoints, indicating that the presence of these signaling factors can be used as an in vitro biomarker of immune suppression/activation (without the need for a mouse). Additional preliminary data from Salus Discovery addresses the second hurdle by demonstrating that the T-CBC assay can be miniaturized onto a microfluidic platform, requiring only 104 to 105 patient cells. By combining microfluidic technology with the T-CBC assay we can decrease the number of cells required per assay while maintaining (and possibly improving, see preliminary data) performance moving the assay towards commercial use within the auto-immunity and transplant fields.